Hypothalamic energy balance gene responses in the Sprague-Dawley rat to supplementation of high-energy diet with liquid ensure and subsequent transfer to chow.

Energy dense, high fat, high sugar, foods and beverages in our diet are a major contributor to the escalating global obesity problem. Here, we examine the physiological and neuroendocrine effects of feeding rats a solid high-energy (HE) diet with or without a liquid supplement (Ensure) and the consequence of subsequently transferring animals back to chow (C). Outbred Sprague-Dawley rats were fed C until 49-56 days of age, and then transferred a HE diet for 3 weeks before allocation to one of two weight-matched groups. Over the next 10 weeks, one group remained on HE diet, whereas the other had access to the liquid diet, chocolate Ensure (EN), in addition to HE diet (HE + EN). Half the rats from each group were then killed, and the remainder were returned to C for 3 weeks. Supplementation of the HE diet with EN accelerated weight gain and increased daily energy intake, adipose tissue mass, and circulating leptin levels. Transferring animals back to C caused a decrease in bodyweight in the HE + EN group, whereas HE animals were weight stable. Both groups also exhibited voluntary hypophagia, although the magnitude and duration of this response was greater in HE + EN animals. The only effect of Ensure on the hypothalamic genes studied was on tyrosine kinase B expression in the ventromedial hypothalamic nucleus (VMH), which was increased in rats given the supplement. Withdrawal of the obesogenic diets decreased gene expression for cocaine-and-amphetamine regulated transcript (CART) and dynorphin (DYN) in the arcuate nucleus (ARC), and DYN and brain-derived neurotrophic factor (BDNF) in the VMH, whereas neuropeptide Y (NPY) gene expression in the ARC was increased. These changes were independent of previous dietary history. EN supplementation generates distinct physiological responses, yet has a minimal effect on hypothalamic neuropeptide or receptor gene expression, possibly due to the development of leptin resistance. Withdrawal of obesogenic diets induces changes in the gene expression consistent with NPY, CART and BDNF attempting to oppose weight gain on either HE or HE + EN.

Introduction of a high-energy diet acutely up-regulates hypothalamic cocaine and amphetamine-regulated transcript, Mc4R and brown adipose tissue uncoupling protein-1 gene expression in male Sprague-Dawley rats.

Obesity is an escalating problem in Western societies. Susceptibility to weight gain within an obesogenic environment is variable. It remains unclear how the range of weight gain responses are generated. It is possible that an individual's immediate and/or sustained appetite for apparently palatable foods, or metabolic adaptations to a new diet could be important. The present study therefore examined the short- to medium-term effects of a high-energy (HE) diet on bodyweight, food intake, and energy balance-related signalling systems. Sprague-Dawley rats were fed either chow or an HE diet for 12 h, 24 h, 48 h or 14 days. Blood hormones and metabolites were assayed, and expression of uncoupling protein-1 (UCP-1) and hypothalamic energy-balance related genes were determined by Northern blotting or in situ hybridisation, respectively. Short-term exposure (12 h, 24 h, 48 h) to the HE diet had no effect on grams of food consumed, but caloric intake was increased. Exposure to HE diet for 14 days (medium term) established a bodyweight differential of 7.7 g, and animals exhibited a transient increase in caloric intake of 5 days duration. Terminal levels of leptin, insulin, glucose and non-esterified fatty acids (NEFAs) were all increased in HE-fed animals. UCP-1 mRNA was elevated in interscapular brown adipose tissue from HE-fed rats only at 12 h. Cocaine and amphetamine-regulated transcript (CART) and Mc4R gene expression in the hypothalamus were increased after 12 h and 24 h on an HE diet, respectively. The rats appear to passively over-consume calories as a result of consuming a similar weight of a more energy dense food. This evokes physiological responses, which adjust caloric intake over several days. Circulating NEFA and insulin concentrations, UCP-1, Mc4R and CART gene expression are increased as an immediate consequence of consuming HE diet, and may be involved in countering hypercaloric intake. Circulating leptin is increased in the HE-fed animals after 48 h, reflecting their increasing adiposity.

Peripherally administered [Nle4,D-Phe7]-alpha-melanocyte stimulating hormone increases resting metabolic rate, while peripheral agouti-related protein has no effect, in wild type C57BL/6 and ob/ob mice.

The melanocortin system coordinates the maintenance of energy balance via the regulation of both food intake and energy expenditure. Leptin, a key adipogenic hormone involved in the regulation of energy balance is thought to act by stimulating production, in the hypothalamic arcuate nucleus, of alpha-melanocyte stimulating hormone (alphaMSH), a potent agonist of MC3/4 melanocortin receptors located in the paraventricular nucleus of the hypothalamus. Additionally leptin inhibits release of agouti-related protein (AgRP), an MC4R antagonist. During periods of caloric restriction, weight loss is not sustained because compensatory mechanisms, such as reduced resting metabolic rate (RMR) are brought into play. Understanding how these compensatory systems operate may provide valuable targets for pharmaceutical therapies to support traditional dieting approaches. As circulating leptin is reduced during caloric restriction, it may mediate some of the observed compensatory responses. In addition to decreases in circulating leptin levels, circulating AgRP is increased during fasting in rodents while alphaMSH is decreased. As central administration of AgRP depresses metabolism, we hypothesised that the peripheral rise in AgRP might be involved in signalling the depression of RMR during food restriction. We hypothesised that changes in plasma AgRP and alphaMSH may coordinate the regulation of changes in energy expenditure acting through central MC4 melanocortin receptors via the sympathetic nervous system.We show here that acute peripherally administered AgRP at supra-physiological concentrations in both lean (C57BL/6) and obese leptin-deficient (ob/ob) mice does not depress RMR, possibly because it crosses the blood-brain barrier very slowly compared with other metabolites. However, in vitro AgRP can decrease leptin secretion, by approximately 40%, from adipocytes into culture medium and may via this axis have an effect on energy metabolism during prolonged caloric restriction. In contrast, peripheral [Nle4,D-Phe7]-alpha MSH produced a large and sustained increase in resting energy expenditure (0.15 ml O2/min; P < 0.05) with a similar response in leptin-deficient ob/ob mice (0.27 ml O2/min) indicating that this effect is independent of the status of leptin production in the periphery. In both cases respiratory exchange ratio and the levels of energy expended on spontaneous physical activity were unaffected by the administration of peripheral [Nle4,D-Phe7]-alpha MSH. In conclusion, alphaMSH analogues that cross the blood-brain barrier may significantly augment dietary restriction strategies by sustaining elevated RMR.

Regulation of adipose tissue leptin secretion by alpha-melanocyte-stimulating hormone and agouti-related protein: further evidence of an interaction between leptin and the melanocortin signalling system.

The central role of the melanocortin system in the regulation of energy balance has been studied in great detail. However, the functions of circulating melanocortins and the roles of their peripheral receptors remain to be elucidated. There is increasing evidence of a peripheral action of melanocortins in the regulation of leptin production by adipocytes. Here we investigate the interaction of alpha-melanocyte stimulating hormone (alpha-MSH) and agouti-related protein (AgRP) in the regulation of leptin secretion from cultured rat adipocytes and examine the changes in circulating alpha-MSH and AgRP in lean and obese rodents after hormonal and energetic challenge. Leptin secretion (measured by ELISA) and gene expression (by real-time quantitative PCR) of differentiated rat adipocytes cultured in vitro were inhibited by the administration of alpha-MSH (EC50=0.24 nM), and this effect was antagonised by antagonists of the melanocortin receptors MC4R and MC3R (AgRP and SHU9119). The presence of MC4R in rat adipocytes (RT-PCR and restriction digest) supports the involvement of this receptor subtype in this interaction. Leptin administered to ob/ob mice in turn increases the release of alpha-MSH into the circulation, suggesting a possible feedback loop between the site of alpha-MSH release and the release of leptin from the adipose tissue. However, the physiological significance of this putative feedback probably depends upon the underlying state of energy balance, since in the fasting state low plasma alpha-MSH is paralleled by low plasma leptin.

The sympathetic nervous system in white adipose tissue regulation.

Sympathetic stimulation has long been recognized to mobilise fatty acids from white adipose tissue. However, it is now apparent that adipose tissue is not only concerned with energy storage as fat, but is a major endocrine and secretory organ. This change has resulted from the identification of leptin as a hormone of energy balance secreted by white adipose tissue. The sympathetic system is a key regulator of leptin production in white fat. Sympathomimetic amines, cold exposure or fasting (which lead to sympathetic stimulation of white fat), decrease ob gene expression in the tissue and leptin production. On the other hand, sympathetic blockade often increases circulating leptin and ob gene expression, and it is postulated that the sympathetic system has a tonic inhibitory action on leptin synthesis. In rodents this action is through stimulation of, beta3-adrenoceptors. The adrenal medulla (as opposed to the direct sympathetic innervation) has been thought to play only a minor role in the catecholaminergic regulation of white adipose tissue. However, in rodents responses of the leptin system to adrenergic blockade vary with the method used. Changes in leptin and ob gene expression are considerably less using methods of blockade that only effect the terminal adrenergic innervation, rather than medullary secretions as well. Stimulation of the leptin system increases sympathetic activity and hence metabolic activity in many tissues. As well as leptin, other (but not all) secretions from white adipose tissue are subject to sympathetic regulation. In obesity the sympathetic sensitivity of adipose tissue is reduced and this factor may underlie the dysregulation of leptin production and other adipose tissue secretions.

Regulation of leptin production: sympathetic nervous system interactions.

Leptin is secreted primarily from white adipose tissue and stimulates long-form OB-Rb receptors in the hypothalamus to decrease food intake and increase energy expenditure. A variety of neuropeptides are involved in these responses, including neuropeptide Y, agouti-related protein, the prepro-melanocortin system and cocaine- and amphetamine-regulated transcript. OB-Rb receptors (and other receptor isoforms) are also found in peripheral tissues. Leptin is now known to have a wide range of peripheral actions and is involved in activating the immune system, haematopoiesis, angiogenesis and as a growth factor, as well as being a regulator of many cellular functions. The identification of leptin has led to reappraisal of the role of white adipose tissue from being an organ concerned primarily with energy storage as fat to an understanding that it is also a major endocrine and secretory organ. While the importance of the sympathetic nervous system in mobilising fatty acids from adipose tissue has long been known, it has become apparent that the sympathetic system is a key regulator of leptin production in white adipose tissue as well. Sympathomimetic amines and cold exposure or fasting (which lead to sympathetic stimulation of white fat), decrease leptin gene expression in the tissue and leptin production. On the other hand, sympathetic blockade often increases circulating leptin and leptin gene expression, and it is possible that the sympathetic system has a tonic inhibitory action on leptin synthesis. Apart from the few instances where leptin is absent, leptin levels are increased in obesity, while the sympathetic sensitivity of adipose tissue is reduced, consistent with the high leptin levels that are seen. The dysregulation of energy balance leading to obesity may partly involve a decrease in leptin sensitivity, or the leptin system may be set to have maximal effects at low leptin levels.

Leptin: fundamental aspects.

The discovery of leptin, the product of the ob gene, has led to major developments in understanding the regulation of energy balance. It is now recognised that leptin is produced in several organs additional to white adipose tissue, including brown fat, the placenta and fetal tissues (such as heart and bone/cartilage). The hormone has multiple functions-in inhibiting food intake, in the stimulation/maintenance of energy expenditure, as a signal to the reproductive system and as a 'metabolic' hormone influencing a range of processes (for example, insulin secretion, lipolysis, sugar transport). The production of leptin by white fat is subject to a number of regulatory influences, including insulin and glucocorticoids (which are stimulatory), and fasting and beta-adrenoceptor agonists (which are inhibitory). A key role in the regulation of leptin production by white fat is envisaged for the sympathetic system, operating through beta3-adrenoceptors. The leptin receptor gene is widely expressed, with the several splice variants exhibiting different patterns of expression. The long form variant (Ob-Rb) is expressed particularly in the hypothalamus, although it is being increasingly identified in other tissues. Leptin exerts its central effects through several neuroendocrine systems, including neuropeptide Y, glucagon-like peptide-1, melanocortins, corticotrophin releasing hormone (CRH) and cocaine- and amphetamine-regulated transcript (CART). In essence, the leptin system now appears highly complex, the hormone being involved in a range of physiological processes in a manner far transcending the initial lipostatic concept. This complexity may reduce the potential of the leptin system as a target for anti-obesity therapy.

Induction of uncoupling protein-2 (UCP2) gene expression on the differentiation of rat preadipocytes to adipocytes in primary culture.

We have examined uncoupling protein-2 (UCP2) gene expression in the adipose tissue of obese and normal rats and mice, and also in differentiated rat adipocytes in primary culture. Expression of the UCP2 gene was examined in rat and mouse adipose tissues using both RT-PCR and Northern blotting. Although the RT-PCR was not quantitative, the band corresponding to the UCP2 mRNA was stronger in white adipose tissue than in brown fat, regardless of the body weight of the rats. In agreement with the RT-PCR data, there was a higher level of UCP2 mRNA in the white adipocytes than in brown adipocytes, the level being greater in obese mice. Fibroblastic preadipocytes were obtained from the inguinal fat pad of suckling rats. Lipid droplets developed inside the cells upon differentiation and adipsin and UCP2 mRNAs were detected by Northern blotting. Both mRNAs were evident in the adipocytes at 4, 6, and 10 d after the induction of differentiation. There was no indication that the expression of UCP2 was markedly affected by the addition of leptin, dexamethasone or isoprenaline.

Hormonal and neuroendocrine regulation of energy balance--the role of leptin.

A new dimension to the regulation of energy balance has come from the identification of the ob (obese) gene and its protein product, leptin. Leptin is produced primarily in white adipose tissue, but synthesis also occurs in brown fat and the placenta. Several physiological functions have been described for leptin the inhibition of food intake, the stimulation/maintenance of energy expenditure, as a signal of energy reserves to the reproductive system, and as a factor in haematopoiesis. The production of leptin by white fat is influenced by a number of factors, including insulin and glucocorticoids (which are stimulatory), and fasting, cold exposure and beta-adrenoceptor agonists (which are inhibitory). A key role in the regulation of leptin production is envisaged for the sympathetic nervous system, operating through beta 3-adrenoceptors. The leptin receptor gene is expressed in a wide range of tissues, and several splice variants are evident. A long form variant (Ob-Rb) with an intracellular signalling domain is found particularly in the hypothalamus. Leptin exerts its central effects through neuropeptide Y, and through the glucagon-like peptide-1 and melanocortin systems, but it may also interact with other neuroendocrine pathways. The role and function of the leptin system in agricultural animals has not been established, but it offers a potential new target for the manipulation of body fat.

Hyperleptinaemia in mice induced by administration of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine.

Alpha-methyl-p-tyrosine (alphaMPT), an inhibitor of tyrosine hydroxylase, was administered to mice to block noradrenaline synthesis. Ten hours after injection of alphaMPT there was a 6-fold increase in plasma leptin. The level of ob mRNA in epididymal white adipose tissue was also increased, but UCP1 mRNA in brown fat fell. In contrast to lean mice, ob mRNA in white fat of ob/ob mice was not increased by alphaMPT. AlphaMPT raised plasma leptin in fasted as well as fed mice. Hyperleptinaemia was attenuated by treatment with a beta3-adrenoceptor agonist. Inhibition of noradrenaline synthesis leads to the rapid induction of hyperleptinaemia; it is suggested that sympathetic tone plays a pivotal role in regulating leptin production.

Postnatal development of the ob gene system: elevated leptin levels in suckling fa/fa rats.

The postnatal development of the ob gene system has been examined in Zucker fa/fa and +/fa plus +/+ (referred to as +/?) rats. White adipose tissue was taken from animals aged 1 to 28 days. Before weaning, white fat was predominantly subcutaneous, the amount increasing rapidly after birth. ob mRNA was detected by Northern blotting in samples of inguinal fat at 1 day of age and thereafter. Circulating leptin, measured by enzyme-linked immunosorbent assay, was also detectable from 1 day of age, the level rising to a peak by 10 days of age and then declining. The fa/fa genotype was determined from the size of the product after Msp I digestion of the Ob-receptor gene obtained by polymerase chain reaction amplification of genomic DNA. No statistically significant difference in ob mRNA level between fa/fa and +/? animals was obtained before 25 days of age. However, leptin levels were significantly higher in the fa/fa mutant by 10 days of age, despite the absence of any significant elevation in the weight of the major fat depots or in ob mRNA level. It is concluded that the ob gene is expressed and leptin produced early in postnatal life in rats; the elevation of circulating leptin in suckling fa/fa animals indicates that dysregulation of the leptin system occurs before the overt development of the obese phenotype.

Localization of leptin receptor mRNA splice variants in murine peripheral tissues by RT-PCR and in situ hybridization.

Expression of leptin receptor splice variants, including the long form variant (Ob-Rb), has been examined in murine peripheral tissues. RT-PCR indicates that the leptin receptor, Ob-R, and in particular the Ob-Ra splice variant are expressed in a wide range of tissues. Expression of the Ob-R receptor was localized by in situ hybridization to specific sites in the spleen, testes, kidney, liver, lung, and adrenal. However, the long form leptin receptor, Ob-Rb, was only expressed at significant levels in the medulla of the adrenal and the inner zone of the medulla of the kidney. The specific sites of expression of different splice variants within peripheral tissues has important implications with regard to the function of leptin.

Regulation of leptin receptor and NPY gene expression in hypothalamus of leptin-treated obese (ob/ob) and cold-exposed lean mice.

Leptin receptor gene expression has been measured in arcuate and ventromedial hypothalamic nuclei. Receptor mRNA in both hypothalamic areas was higher in obese mice than in lean littermates. Twice daily leptin administration for 7 days profoundly affected food intake, reduced leptin receptor mRNA in the arcuate nucleus, and had a similar effect on neuropeptide Y gene expression. A single leptin injection was ineffective. Exposure of lean mice to cold for 24 h caused an induction of leptin receptor and NPY mRNA which was normalized when animals were returned to the warm. Regulation of receptor gene expression may be an important component in the reading of the leptin signal.

Rapid inhibition of ob gene expression and circulating leptin levels in lean mice by the beta 3-adrenoceptor agonists BRL 35135A and ZD2079.

The acute effect of two selective beta 3-adrenoceptor agonists, BRL 35135A and ZD2079, on the expression of the ob gene and plasma leptin levels has been examined in mice. By 4-5 h after the administration of either beta 3-agonist to lean animals there was a major loss of ob mRNA from epididymal white adipose tissue. This was accompanied by a substantial fall in circulating leptin levels, as measured by an ELISA. Even 24 h after the first administration of beta 3-agonists, ob mRNA levels and circulating leptin levels remained low. In contrast to lean animals, treatment with BRL 35135A had only a minor effect on ob mRNA levels in obese (ob/ob) mice. Regulation of leptin production appears to involve a negative feedback loop to white adipose tissue through the sympathetic nervous system suppressing ob gene transcription via beta 3-adrenoceptors; an impairment in this loop is evident in the ob/ob mutant.

Circulating leptin levels are modulated by fasting, cold exposure and insulin administration in lean but not Zucker (fa/fa) rats as measured by ELISA.

The development of a sensitive enzyme-linked immunosorbent assay (ELISA) is described for the quantitation of plasma leptin levels in both mice and rats. Approximately 1.5 ng leptin/ml plasma was detected in lean mice but significantly (p < 0.01) less was found in the plasma of ob/ob mice. In lean Zucker rats leptin circulated at approximately 4ng/ml plasma whereas levels were elevated more than 6-fold in fa/fa rats. Circulating leptin levels declined (p < 0.05) in lean rats which were fasted or exposed to 4 degrees C for 24h, but subsequently recovered to normal within 12h of refeeding or warming, respectively. Administration of insulin increased leptin levels in lean rats within 4h (p < 0.01). However, leptin levels were unchanged in fa/fa rats exposed to the same physiological stimuli.